The goal of this project is to evaluate the role of the autonomic neurotransmitters in regulation, and possibly the maintenance, of myocardial rhythmicity. Whole hearts from developing chick embryos, or cell cultures prepared from such hearts, are investigated for the presence of functioning neurotransmitter systems. Such systems must be considered to be extraneuronal in aneural myocardial cell cultures and in hearts of developing chick embryos prior to morphologic and functional innervation. We have demonstrated the components of a cholinergic system in such preparations: (1) a choline uptake mechanism is functional in cultured heart cells, (2) choline acetyltransferase (CAT), the enzyme which catalyzes the synthesis of acetylcholine (Ach) is present in hearts of developing embryos from the third day of incubation (functional cholinergic innervation occurs on day 12 of incubation). CAT has not yet been demonstrated in cultured cells, but the search is continuing. Studies will continue with the study of Ach synthesis and turnover rates in embryos using a sensitive gas-liquid radiochromatographic assay for choline and Ach. The presence of Ach turnover in aneural myocardial tissue would indicate a functional role for this substance. We have already shown that Ach will increase spontaneity and contractile frequency in myocardial cell cultures. Other studies are aimed at the isolation and purification of choline acetyltransferase from non-neural (placenta) and neuronal (brain) sources and to compare their function and control mechanisms. Techniques involving x-ray analysis, substrate and inhibitor affinities will be utilized to find essential functional differences between the two types of enzyme.